Plant lipids have a variety of industrial and nutritional uses and are central to plant membrane function and climatic adaptation. These lipids represent a vast array of chemical structures, and these structures determine the physiological and industrial properties of the lipid. Many of these structures result either directly or indirectly from metabolic processes that alter the degree of unsaturation of the lipid. Different metabolic regimes in different plants produce these altered lipids, and either domestication of exotic plant species or modification of agronomically adapted species is usually required to produce economically large amounts of the desired lipid.
There are serious limitations to using mutagenesis to alter fatty acid composition and content. Screens will rarely uncover mutations that a) result in a dominant (“gain-of-function”) phenotype, b) are in genes that are essential for plant growth, and c) are in an enzyme that is not rate-limiting and that is encoded by more than one gene. In cases where desired phenotypes are available in mutant crop lines, their introgression into elite lines by traditional breeding techniques is slow and expensive, since the desired oil compositions are likely the result of several recessive genes.
Recent molecular and cellular biology techniques offer the potential for overcoming some of the limitations of the mutagenesis approach, including the need for extensive breeding. Some of the particularly useful technologies are seed-specific expression of foreign genes in transgenic plants [see Goldberg et al. (1989) Cell 56:149-160], and the use of antisense RNA to inhibit plant target genes in a dominant and tissue-specific manner [see van der Krol et al. (1988) Gene 72:45-50]. Other advances include the transfer of foreign genes into elite commercial varieties of commercial oilcrops, such as soybean [Chee et al (1989) Plant Physiol. 91:1212-1218; Christou et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:7500-7504; Hinchee et al. (1988) Bio/Technology 6:915-922; EPO publication 0 301 749 A2], rapeseed [De Block et al. (1989) Plant Physiol. 91:694-701], and sunflower [Everett et al. (1987) Bio/Technology 5:1201-1204], and the use of genes as restriction fragment length polymorphism (RFLP) markers in a breeding program, which makes introgression of recessive traits into elite lines rapid and less expensive [Tanksley et al. (1989) Bio/Technology 7:257-264]. However, application of each of these technologies requires identification and isolation of commercially-important genes.
Glycerophospholipids in biological membranes are metabolically active and participate in a series of deacylation-reacylation reactions, which may lead to accumulation of polyunsaturated fatty acids (PUFAs) at the sn-2 position of the glycerol backbone. The reacylation reaction is believed to be catalyzed by Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT)), which catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce Phosphatidylcholine (PC) and CoA. LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed triacylglycerol (TAG) composition. LPCAT activity is associated with two structurally distinct protein families, wherein one belongs to the Lysophosphatidic acid acyltransferase (LPAAT) family of proteins and the other belongs to the membrane bound O-acyltransferase [“MBOAT”] family of proteins. In yeast, YOR175c, an acyltransferase belonging to the MBOAT family of proteins, has recently been shown to represent a major acyl-CoA dependent lysophospholipid acyltransferase (Wayne et al.; JBC, 2007, 282:28344-28352). It further was shown by Sandro Sonnino (FEBS Letters, 2007, 581:5511-5516) that the yeast acyiglycerol acyltransferase LCA1 (YOR175c) is a key component of the Lands cycle for phosphatidylcholine turnover.
Stanford et al. (JBC, 2007, 282:30562-30569) found that in yeast the LTP1 gene encodes for an acyltransferase that uses a variety of lysophospholipid species. Together with Slc1, Lpt1p seems to mediate the incorporation of unsaturated acyl chains into the sn-2 position of phospholipids.
Benghezal et al. (JBC, 2007, 282:30845-30855) show that Slc1p and Slc4p appear to be active not only as 1-acylglycerol-3-phosphate O-acyltransferases but also appear to be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.
A newly discovered human LPCAT (LPCAT3), which has distinct substrate preferences, has been identified (Kazachkov et al., Lipids, 2008, 43:895-902). Kazachkov et al. suggest that LPCAT3 is involved in phospholipids remodeling to achieve appropriate membrane lipid fatty acid composition.
Four human MBOATs have been expressed in yeast and two of them, MBOAT5 and MBOAT7 have been implicated in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils (Gijon et al., JBC, 2008, 283:30235-30245).
Altogether more than 300 different fatty acids are known to occur in seed TG. Chain length may range from less than 8 to over 22 carbons. The position and number of double bonds may also be unusual, and hydroxyl, epoxy, or other functional groups can modify the acyl chain. The special physical and chemical properties of the unusual plant fatty acids have been exploited for centuries. Approximately one-third of all vegetable oil is used for non-food purposes. The ability to transfer genes for unusual fatty acid production from exotic wild species to high yielding oilcrops is now providing, for example, the ability to produce new renewable agricultural products (Biochemistry of lipids, lipoproteins and membranes, ed. D. E. Vance and J. Vance, 1996 Elsevier Science).
Given the acyl-editing activity of the MBOAT protein family of genes, it is of interest to find other plant homologs with similar activities and characterize the effect of their expression on seed oil composition.
The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.
The following sequences comply with 37 C.F.R. §§1.821-1.825 (“Requirements for patent applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
SEQ ID NO:1 corresponds to the cDNA insert sequence from esc1c.pk007.c17 (CoMBOAT).
SEQ ID NO:2 corresponds to the ORF encoded by SEQ ID NO:1.
SEQ ID NO:3 corresponds to the amino acid sequence encode by SEQ ID NO:2.
SEQ ID NO:4 corresponds to the amino acid sequence of the MBOAT family protein from Vitis vinifera (GI:225426775).
SEQ ID NO:5 corresponds to the amino acid sequence of the MBOAT family protein from Arebidopsis thaliana (GI:22329514).
SEQ ID NO:6 corresponds to cDNA insert sequence from fds1n.pk001.k4 (McMBOAT).
SEQ ID NO:7 corresponds to the McLPCAT 5Race primer.
SEQ ID NO:8 corresponds to the McLPCATnew1 primer.
SEQ ID NO:9 corresponds to the McMBOAT 5′RACE sequence.
SEQ ID NO:10 corresponds to the McMBOAT full cDNA sequence.
SEQ ID NO:11 corresponds to the ORF encoded by SEQ ID NO:10.
SEQ ID NO:12 corresponds to the amino acid sequence encoded by SEQ ID NO:11.
SEQ ID NO:13 corresponds to the cDNA insert sequence from esc1c.pk002. d16 (CoDGAT2).
SEQ ID NO:14 corresponds to the ORF encoded by SEQ ID NO:13.
SEQ ID NO:15 corresponds to the amino acid sequence encoded by SEQ ID NO:14.
SEQ ID NO:16 corresponds to the hypothetical protein from Vitis vinifera (GI:225431649).
SEQ ID NO:17 corresponds to the amino acid sequence of diacylglycerol acyltransferase from Elaeis oleifera. 
SEQ ID NO:18 corresponds to the DNA insert sequence from fds.pk0003.97 (McDGAT2).
SEQ ID NO:19 corresponds to the McDGAT2 Race1 primer.
SEQ ID NO:20 corresponds to the McDGAT2 5′Race sequence.
SEQ ID NO:21 corresponds to the McDGAT2 Not5 primer.
SEQ ID NO:22 corresponds to the McDGAT2 Not3 primer.
SEQ ID NO:23 corresponds to the McDGAT2 sequence flanked by NotI sites.
SEQ ID NO:24 corresponds to the full McDGAT2 cDNA sequence.
SEQ ID NO:25 corresponds to the ORF encoded by SEQ ID NO:24.
SEQ ID NO:26 corresponds to the amino acid sequence encoded by SEQ ID NO:25.
SEQ ID NO:27 corresponds to diacylglycerol acyltransferase from Arabidopsis thaliana. 
SEQ ID NO:28 corresponds to the nucleotide sequence of vector pHD40.
SEQ ID NO:29 corresponds to the nucleotide sequence of vector pKR1543.
SEQ ID NO:30 corresponds to the gene coding sequence of the Momordica charantia conjugase (McConj).
SEQ ID NO:31 corresponds to the nucleotide sequence of vector pKR458.
SEQ ID NO:32 corresponds to the McLPCATNOt5 primer.
SEQ ID NO:33 corresponds to the McLPCATNot3 primer.
SEQ ID NO:34 corresponds to the nucleotide sequence of vector pHD41.
SEQ ID NO:35 corresponds to the nucleotide sequence of vector pKR1548.
SEQ ID NO:36 corresponds to the nucleotide sequence of vector pKR1556.
SEQ ID NO:37 corresponds to the nucleotide sequence of vector pKR1562.
SEQ ID NO:38 corresponds to the CoDGAT-5Not primer.
SEQ ID NO:39 corresponds to the CoDGAT-3Not primer.
SEQ ID NO:40 corresponds to the nucleotide sequence of vector pKR1493.
SEQ ID NO:41 corresponds to the nucleotide sequence of the ORF of Calendula officinalis fatty acid conjugase (CoConj).
SEQ ID NO:42 corresponds to the nucleotide sequence of vector pKR1487.
SEQ ID NO:43 corresponds to the CoLPCAT-5Not primer.
SEQ ID NO:44 corresponds to the CoLPCATNco-3 primer.
SEQ ID NO:45 corresponds to the CoLPCATNco-5 primer.
SEQ ID NO:46 corresponds to the CoLPCAT-3Not primer.
SEQ ID NO:47 corresponds to the nucleotide sequence of CoMBOAT with the NcoI site removed.
SEQ ID NO:48 corresponds to the nucleotide sequence of vector pLF166.
SEQ ID NO:49 corresponds to the nucleotide sequence of vector pKR1492.
SEQ ID NO:50 corresponds to the nucleotide sequence of vector pKR1498.
SEQ ID NO:51 corresponds to the nucleotide sequence of vector pKR1504.
SEQ ID NO:52 corresponds to the nucleotide sequence of vector pKR539.
SEQ ID NO:53 corresponds to the nucleotide sequence of vector pKR1563.
SEQ ID NO:54 corresponds to the nucleotide sequence of vector pKR1564.
SEQ ID NO:55 corresponds to the nucleotide sequence of vector pKR1565.
SEQ ID NO:56 corresponds to the nucleotide sequence of vector pKR1507.
SEQ ID NO:57 corresponds to the nucleotide sequence of vector pKR1508.
SEQ ID NO:58 corresponds to the nucleotide sequence of vector pKR1509.
SEQ ID NO:59 corresponds to the nucleotide sequence of vector pKR1510.
SEQ ID NO:60 corresponds to the nucleotide sequence of vector pKR1561.
SEQ ID NO:61 corresponds to the nucleotide sequence of vector pKR1544.
SEQ ID NO:62 corresponds to the nucleotide sequence of vector pKR1549.
SEQ ID NO:63 corresponds to the nucleotide sequence of vector pKR1546.
SEQ ID NO:64 corresponds to the nucleotide sequence of vector pKR1557.
SEQ ID NO:65 corresponds to the nucleotide sequence of vector pKR1560.
SEQ ID NO:66 corresponds to the nucleotide sequence of vector pKR1545.
SEQ ID NO:67 corresponds to the nucleotide sequence of vector pKR1550.
SEQ ID NO:68 corresponds to the nucleotide sequence of vector pKR1547.
SEQ ID NO:69 corresponds to the nucleotide sequence of vector pKR1558.
SEQ ID NO:70 corresponds to the nucleotide sequence of vector pKR1559.
SEQ ID NO:71 corresponds to the nucleotide sequence of vector pKR1552.
SEQ ID NO:72 corresponds to the nucleotide sequence of vector pKR1554.
SEQ ID NO:73 corresponds to the nucleotide sequence of vector pKR1022.
SEQ ID NO:74 corresponds to the nucleotide sequence of vector pKR1553.
SEQ ID NO:75 corresponds to the nucleotide sequence of vector pKR1555.
SEQ ID NO:76 corresponds to the nucleotide sequence of vector pLF167.
SEQ ID NO:77 corresponds to the nucleotide sequence encoding the fatty acid desaturase (nt1-nt 1149 (STOP)) from Vernonia galamensis. 
SEQ ID NO:78 corresponds to the amino acid sequence encoded by SEQ ID NO:77.
SEQ ID NO:79 corresponds to the nucleotide sequence encoding an epoxidase from Vernonia galamensis. 
SEQ ID NO:80 corresponds to the amino acid sequence encoded by SEQ ID NO:79.
SEQ ID NO:81 corresponds to the nucleotide sequence encoding the delta-5 acyl-CoA desaturase from Limnanthes alba. 
SEQ ID NO:82 corresponds to the amino acid sequence encoded by SEQ ID NO:81.
SEQ ID NO:83 corresponds to the nucleotide sequence encoding the fatty acyl-CoA elongase from Limnanthes alba. 
SEQ ID NO:84 corresponds to the amino acid sequence encoded by SEQ ID NO:83.
SEQ ID NO:85 corresponds to the nucleotide sequence encoding the a conjugase from Impatiens balsamina. 
SEQ ID NO:86 corresponds to the amino acid sequence encoded by SEQ ID NO:85.
SEQ ID NO:87 corresponds to the nucleotide sequence encoding a conjugase from Momordica charantia. 
SEQ ID NO:88 corresponds to the amino acid sequence encoded by SEQ ID NO:87.
SEQ ID NO:89 corresponds to the nucleotide sequence encoding a conjugase from Chrysobalanus icaco. 
SEQ ID NO:90 corresponds to the amino acid sequence encoded by SEQ ID NO:89.
SEQ ID NO:91 corresponds to the nucleotide sequence encoding a conjugase from Licania michauxii. 
SEQ ID NO:92 corresponds to the amino acid sequence encoded by SEQ ID NO:91.
SEQ ID NO:93 corresponds to the nucleotide sequence encoding a conjugase from Aleurites fordii. 
SEQ ID NO:94 corresponds to the amino acid sequence encoded by SEQ ID NO:93.
SEQ ID NO:95 corresponds to the nucleotide sequence encoding a Class II conjugase from Aleurites fordii. 
SEQ ID NO:96 corresponds to the amino acid sequence encoded by SEQ ID NO:95.
SEQ ID NO:97 corresponds to the amino acid sequence from the hydroxylase from Ricinus communis. 
SEQ ID NO:98 corresponds to the nucleotide sequence of a conjugase from Calendula officialis. 
SEQ ID NO:99 corresponds to the amino acid sequence encoded by SEQ ID NO:98.
SEQ ID NO:100 corresponds to the nucleotide sequence of a conjugase from Calendula officialis. 
SEQ ID NO:101 corresponds to the amino acid sequence encoded by SEQ ID NO:100.
SEQ ID NO:102 corresponds to the nucleotide sequence of a conjugase from Dimorphotheca sinuata. 
SEQ ID NO:103 corresponds to the amino acid sequence encoded by SEQ ID NO:102.
SEQ ID NO:104 corresponds to the nucleotide sequence of a conjugase from Dimorphotheca sinuata. 
SEQ ID NO:105 corresponds to the amino acid sequence encoded by SEQ ID NO:104.
SEQ ID NO:106 corresponds to the nucleotide sequence of vector pKR272.
SEQ ID NO:107 corresponds to the nucleotide sequence of vector pKR278.
SEQ ID NO:108 corresponds to the forward primer RcHydroxy-5.
SEQ ID NO:109 corresponds to the reverse primer RcHydroxy-3.
SEQ ID NO:110 corresponds to the nucleotide sequence of vector pLF241.
SEQ ID NO:111 corresponds to the nucleotide sequence of vector pKR1687.
SEQ ID NO:112 corresponds to the nucleotide sequence of vector pKR1742.
SEQ ID NO:113 corresponds to the nucleotide sequence of vector pKR1733.
SEQ ID NO:114 corresponds to the nucleotide sequence of vector pKR1745.
SEQ ID NO:115 corresponds to the nucleotide sequence of vector pKR966.
SEQ ID NO:116 corresponds to the nucleotide sequence of vector pKR1542.
SEQ ID NO:117 corresponds to the nucleotide sequence of vector pKR1743.
SEQ ID NO:118 corresponds to the nucleotide sequence of vector pKR1734.
SEQ ID NO:119 corresponds to the nucleotide sequence of vector pKR1746.
SEQ ID NO:120 corresponds to the GmMBOAT1 genomic sequence.
SEQ ID NO:121 corresponds to the GmMBOAT1 coding sequence.
SEQ ID NO:122 corresponds to the GmMBOAT1 amino acid sequence.
SEQ ID NO:123 corresponds to the GmMBOAT2 genomic sequence.
SEQ ID NO:124 corresponds to the GmMBOAT2 coding sequence.
SEQ ID NO:125 corresponds to the GmMBOAT2 amino acid sequence.
SEQ ID NO:126 corresponds to the GmLPCAT1-5 primer.
SEQ ID NO:127 corresponds to the GmLPCAT1-3 primer.
SEQ ID NO:128 corresponds to the nucleotide sequence of vector pLF164.
SEQ ID NO:129 corresponds to the GmLPCAT2-5 primer.
SEQ ID NO:130 corresponds to the nucleotide sequence of vector pLF165.
SEQ ID NO:131 corresponds to the nucleotide sequence of vector pKR1813.
SEQ ID NO:132 corresponds to the nucleotide sequence of vector pKR1814.
SEQ ID NO:133 corresponds to the nucleotide sequence of vector pKR1821.
SEQ ID NO:134 corresponds to the nucleotide sequence of vector pKR1822.
SEQ ID NO:135 corresponds to the cDNA insert sequence from eel1c.pk002.h9 (EuphMBOAT).
SEQ ID NO:136 corresponds to the ORF encoded by SEQ ID NO:135.
SEQ ID NO:137 corresponds to the amino acid sequence encoded by SEQ ID NO:136.
SEQ ID NO:138 corresponds to the EIMBOAT-5Not primer.
SEQ ID NO:139 corresponds to the oEU mb-2 primer.
SEQ ID NO:140 corresponds to the nucleotide sequence of vector pKR1823.
SEQ ID NO:141 corresponds to the nucleotide sequence of vector pKR1827.
SEQ ID NO:142 corresponds to the nucleotide sequence of vector pKR1836.
SEQ ID NO:143 corresponds to the nucleotide sequence of vector pKR1815.
SEQ ID NO:144 corresponds to the nucleotide sequence of vector pKR1835.
SEQ ID NO:145 corresponds to the nucleotide sequence of vector pKR1203.
SEQ ID NO:146 corresponds to the nucleotide sequence of vector pHD1.
SEQ ID NO:147 corresponds to the nucleotide sequence of vector pKR1645.
SEQ ID NO:148 corresponds to the nucleotide sequence of vector pKR1646.
SEQ ID NO:149 corresponds to the nucleotide sequence of vector pKR1649.
SEQ ID NO:150 corresponds to the nucleotide sequence of vector pKR1650.
SEQ ID NO:151 corresponds to the nucleotide sequence of vector pKR1818.
SEQ ID NO:152 corresponds to the nucleotide sequence of vector pKR1826,
SEQ ID NO:153 corresponds to the nucleotide sequence of vector pKR1844.
SEQ ID NO:154 corresponds to the nucleotide sequence of vector pKR1671.
SEQ ID NO:155 corresponds to the nucleotide sequence of vector pKR1672.
SEQ ID NO:156 corresponds to the nucleotide sequence of vector pKR1673.
SEQ ID NO:157 corresponds to the nucleotide sequence of vector pKR1674.
SEQ ID NO:158 corresponds to the nucleotide sequence of vector pKR1845.